E2 proteins: modulators of papillomavirus transcription and replication.
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Animals↗
Biomedical subjects
G Steger
Publications and source records attributed to G Steger.
High resolution SSCP by optimization of the temperature by transverse TGGE.
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Base Sequence↗
Interdependence of pathogenicity and replicability with potato spindle tuber viroid.
After the unexpected appearance of lethal symptoms on tomato plants infected with the PSTVd strain Intermediate Di, viroids were isolated and sequenced. It was found that a new strain, named RG 1, had been generated spontaneously in our greenhouse. In a different series of plant passages two new strains, named QF A and QF B, were detected which coexisted with the wild-type strain Di. Strains QF A and QF B showed intermediate symptoms when inoculated separately. In order to confirm the working hypothesis that the more pathogenic strain outcompetes the less pathogenic strain but strains of similar pathogenicity might coexist in the host, strains of different pathogenicity were mixed for inoculation in a ratio from 1:1 to 1:100 (more pathogenic:less pathogenic). The concentrations of the individual strains were determined 6 weeks postinfection with the method of nondenaturing polyacrylamide gel electrophoresis, and the working hypothesis was confirmed. The total concentrations of viroids in infected plants were very similar, irrespective of whether severe, intermediate, or mild strains or mixtures of different strains were present. The mutations in all new strains (3 in RG 1, 2 in QF A, 3 in QF B) were located in the so-called virulence-modulating region. The mutations of strain RG 1 influenced dramatically the thermodynamic stability of the native rod-like structure, as determined experimentally by temperature-gradient gel electrophoresis. Since during replication a multihairpin structure is generated transiently which is transformed afterwards into the rod-like structure, a lower thermodynamic stability of the rod-like structure leads to a higher accumulation of the transient structure. It is assumed that the transient structure, which is active in replication as shown earlier, is essential also in pathogenesis. This model explains the experimentally determined interdependence between pathogenicity and replicability of PSTVd strains.
Base Sequence↗
The bovine papillomavirus 1 E2 protein contains two activation domains: one that interacts with TBP and another that functions after TBP binding.
The E2 transactivator of bovine papillomavirus type-1 is unable to activate minimal promoters in vivo that contain only E2 binding sites and a TATA box. This block can be overcome by over-expression of human TATA binding protein (TBP) or by the addition of either SP1 binding sites or an initiator element to the promoter, suggesting that the binding of TFIID may normally be a rate-limiting step for activation by E2. Surprisingly, purified E2 and TBP bind co-operatively to DNA in vitro when the sites are closely spaced. E2 does not affect the on rate of association but reduces the off rate. The E2 region responsible for this effect is located in the hinge region that links the classic transactivation and DNA binding domains. We demonstrate that the TBP stabilizing domain contributes in vivo to co-operativity with co-expressed TBP and to activation of the major late minimal promoter (MLP) containing E2 sites. In contrast, promoters with SP1 sites are activated to wild-type levels by such a mutant. This promoter specificity is also evident in vitro. A truncated E2 mutant, lacking the classic transactivation domain but containing the TBP stabilizing domain, stimulates transcription of the MLP in vitro, but does not activate promoters with SP1 sites. In conclusion, our results show that the E2 transactivation domain has a modular structure. We have identified one domain which probably acts at an early step in the assembly of the pre-initiation complex and which is involved in reducing the dissociation rate of bound TBP in vitro. The classic N-terminal activation domain of E2 might affect one or several step(s) in the assembly of the preinitiation complex occurring after the binding of TFIID.
Adenoviridae↗
Randomised phase II study of epirubicin-vindesine versus mitoxantrone-vindesine in metastatic breast cancer.
The purpose of this study was to compare the activity and toxicity of epirubicin-vindesine (EV) with mitoxantrone-vindesine (MV) in patients with metastatic breast cancer. A total of 295 patients was randomly allocated to treatment with vindesine 3 mg/m2 combined with either epirubicin 40 mg/m2 or mitoxantrone 10 mg/m2. All drugs were given by intravenous push, treatment cycles were repeated at 3-4 week intervals. 255 patients were available for response, and 283 for toxicity. EV and MV yielded similar objective response rates (34 and 26%, respectively), response durations, times to progression and survival. Median time to remission was 1.8 and 3.1 months (P = 0.006) with EV and MV, respectively. In patients with visceral metastases, response rate was higher with EV than MV (40 versus 23%; P = 0.03). Patients receiving MV had less nausea/vomiting (P = 0.007) and alopecia (P = < 0.001) of WHO grade > or = 2. Bone marrow, cardiac and other toxicities were mild with both treatments. The observed differences in activity and toxicity between the two regimens appear to have clinical relevance. EV proved to be more active in visceral disease and to be able to induce remissions more rapidly. Accordingly, patients with visceral metastases or severe tumour-related symptoms may benefit from epirubicin-based treatment. Subjective toxicities, i.e. nausea/vomiting and alopecia, were less frequent and severe with MV. Thus, MV may prove useful in patients with more indolent disease and appears to warrant phase III evaluation in such patients.
Adult↗
A stable hammerhead structure is not required for endonucleolytic activity of a ribozyme in vivo.
Cleavage of a specific target, the mRNA encoding the bacterial neomycin phosphotransferase, by mutant satellite RNA of subterranean clover mottle virus (sSCMoV) ribozymes (Rz) was used to study the role of the hammerhead (Hh) structure in Rz activity in cis and in trans. The bimolecular Rz-target RNA interaction was predicted by computer secondary structure analysis. In vivo, endonucleolytic cleavage was determined in plant protoplasts and compared with in vitro results. Two point mutations within the Hh were studied in detail. A Rz mutant with a point mutation in the most distal nucleotide of the catalytic domain (A14G) showed no endonucleolytic activity in vivo. A second point mutation inside helix II (G11.3C) which destabilizes the helix and, according to thermodynamic calculations, should disrupt the conserved Hh structure, unexpectedly displayed Rz activity in trans in vivo. In vitro, this mutant exhibited an activity similar to the wild-type Rz in cis, but no significant activity in trans. It therefore appears that helix II within the Rz Hh structure is not required in vivo for endonucleolytic activity, nor for stability of the Rz transcript, and that in vitro results are inadequate to predict Rz activity in living cells.
Amino Acid Sequence↗
Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction.
A program is described which calculates the thermal stability and the denaturation behaviour of double-stranded DNAs and RNAs up to a length of 1000 base pairs. The algorithm is based on recursive generation of conditional and a priori probabilities for base stacking. Output of the program may be compared directly to experimental results; thus the program may be used to optimize the nucleic acid fragments, the primers and the experimental conditions prior to experiments like polymerase chain reactions, temperature-gradient gel electrophoresis, denaturing-gradient gel electrophoresis and hybridizations. The program is available in three versions; the first version runs interactively on VAXstations producing graphics output directly, the second is implemented as part of the HUSAR package at GENIUSnet, the third runs on any computer producing text output which serves as input to available graphics programs.
Bacteriophage T7↗
Cooperativity in vivo between the E2 transactivator and the TATA box binding protein depends on core promoter structure.
The E2 transactivator protein of bovine papillomavirus 1 (BPV-1) can strongly stimulate complex promoters such as that of the herpes simplex virus thymidine kinase gene but does not efficiently activate minimal promoters that only contain E2 binding sites and a TATA box. Here we show that overexpression of the human, but not yeast, TATA box binding protein (TBP) in transfection experiments overcomes this block and enables E2 to activate a minimal TATA box-containing promoter. This suggests that recruitment of the TFIID complex to such promoters is normally a rate limiting step for transcriptional activation by E2 in vivo. In contrast, minimal promoters that contain an initiator element in addition to a TATA box are efficiently activated by E2 on its own and this activation is only moderately enhanced by TBP overexpression. In such E2-responsive promoters the TATA box or initiator can be functionally replaced by SP1 binding sites. Both the initiator binding protein, TFII-I, and SP1 have been found to interact physically with components of the TFIID complex. Since either TBP overexpression or the presence of an initiator or SP1 binding sites can increase activation by E2, it seems likely that the principal role of the E2 activation domain is to affect a step in the formation of the transcription initiation complex that occurs after TFIID has bound to the promoter. Sequential action of transcription factors, such as TFII-I, SP1 and E2, may be one type of mechanism underlying the widely observed phenomenon of transcriptional synergy.
Adenoviridae↗
Soluble CD23 reliably reflects disease activity in B-cell chronic lymphocytic leukemia.
PURPOSE: This study was initiated to evaluate whether soluble CD23 (sCD23) reflects disease activity and tumor load in B-cell chronic lymphocytic leukemia (B-CLL) and to determine its prognostic potential for this disease. PATIENTS AND METHODS: The concentration of sCD23 was measured in the serum of 45 B-CLL patients, 50 patients with other lymphoproliferative disorders, and 41 healthy donors (HD). sCD23 serum levels from B-CLL patients were correlated with parameters of disease activity and total tumor mass (TTM) score. In selected cases, sCD23 was measured repeatedly over a 24-month period. Expression, density, and calculated total amount of membrane CD23 on peripheral-blood B-CLL cells, as well as its correlation to sCD23 levels in serum and supernatants, were determined. RESULTS: sCD23 in B-CLL patients serum was highly elevated as compared with other lymphoproliferative disorders, with the exception of immunocytoma (IC). Both advanced Rai stages and active forms of B-CLL were associated with higher levels of sCD23. There was a highly significant reciprocal relationship between sCD23 and lymphocyte count doubling time (LCDT). Serum sCD23 correlated positively with serum deoxythymidine kinase activity and TTM score, but not with absolute lymphocyte counts. The repetitive measurement of serum sCD23 showed the usefulness of this marker in monitoring disease progression in B-CLL. The total amount of membrane CD23 on in vitro-cultured B-CLL cells correlated significantly with sCD23 levels in the supernatant, whereas correlation between serum sCD23 and membrane CD23 on freshly isolated B-CLL cells was absent. CONCLUSION: Our results indicate that sCD23 is a highly sensitive and specific parameter with prognostic potential for B-CLL, which may be used as a tumor marker and may help to assess disease activity.
Biomarkers, Tumor↗
[Primary (preoperative, neoadjuvant) chemotherapy of colon cancer--a therapeutic alternative?].
Primary chemotherapy is an established treatment in selected patients with osteosarcoma and locally advanced breast cancer. In several other tumor entities this therapeutic approach is under clinical investigation. In contrast, colon carcinoma has been believed to be chemoresistant for a long period of time. Thus, no therapeutic approaches dealing with preoperative therapy have been initiated yet. Recent studies showing remission rates as high as 40% in advanced colon cancer and the proof of efficacy for postoperative adjuvant chemotherapy must now lead to reevaluation of the therapeutic approach to this tumor entity. Data from animal models as well as several tumor biologic hypotheses also point to a possible advantage for preoperative therapy in order to ameliorate relapse-free survival and overall survival in these patients. In this work we discuss potential advantages and disadvantages of primary, neoadjuvant strategies of treatment for colon cancer. Based on these pros and cons, clinical studies for a preoperative therapeutic approach appear to be justified and necessary in patients with locally advanced disease and in patients with metastases at the time of diagnosis.
Antineoplastic Agents↗
Relation between skin cancer, humoral responses to human papillomaviruses, and HLA class II molecules in renal transplant recipients.
Human papillomaviruses (HPV), especially the epidermodysplasia verruciformis (EV)-associated HPV 5, 8, 14, 17, 20, and 47, are thought to play a role in the pathogenesis of some skin cancers in recipients of renal allografts. MHC class I and class II genes are involved in the cellular immune response to viral and tumor Ag. Little is known about humoral responses to HPV in recipients with and without skin cancer. We investigated the prevalence of antibodies to the early (E) protein E7 and the major capsid late (L) protein L1 of HPV 8. In addition, we studied the association of HLA class II molecules with these antibody responses. The E7 and L1 open reading frames of HPV 8 were bacterially expressed as beta-galactosidase fusion proteins, which were purified by preparative gel electrophoresis. Serum samples from 36 renal transplant recipients with and 91 recipients without skin cancer were screened for the presence of IgG and IgM antibodies to HPV 8 E7 and L1, by Western blot analysis. The detection of anti-HPV 8 L1 antibodies represents the immune response to HPV 8 and possibly other EV-associated HPV, because cross-reactivity between the representatives of this HPV subgenus can occur. The antibody responses to HLA Ag were used as controls. Recipients who had IgM antibodies but no IgG antibodies to L1 of HPV 8 (patients with no apparent class switch from IgM to IgG) had skin cancer in 50% of cases, whereas recipients who produced IgG antibodies (patients with an apparently good humoral response to L1 of HPV 8) had skin cancer in only 18% of cases. The estimated relative risk of skin cancer in recipients with no class switch, compared with the risk in those with a good humoral response, was 4.5 (95% confidence interval, 1.1 to 18.1). We found no association between the antibody response to HLA Ag and the occurrence of skin cancer. A strong linkage between the absent class switch of antibody production in response to L1 of HPV 8 and HLA-DR7 was observed (relative risk, 26.2). Renal transplant recipients who have no apparent class switch from IgM to IgG production in response to Ag encoded by L1 of HPV 8 or possibly other EV-associated HPV are at an increased risk of skin cancer. The association with HLA-DR7 indicates a genetic control of skin cancer development or regression, involving genes in the class II region of the MHC.
Amino Acid Sequence↗
Multiple pathways of reversion in viroids for conservation of structural elements.
From site-directed mutagenesis of potato spindle tuber viroid (PSTVd) it had been concluded earlier that the formation of a thermodynamically metastable structure containing hairpin II (HP II) is critical for infectivity. In order to differentiate between structural and sequence effects, in the present work base pairs in HP II were exchanged by site-directed double mutations without significant alterations in the native rod-like structure of PSTVd. The mutants were viable and genetically stable in the first generation, but one of the two mutations reverted to the wild-type nucleotide in the second generation. Single-site mutations in the stem of HP II, which had been described as revertants to the wild-type sequence earlier, were analysed with respect to the time course of reversion and the sequence variation during reversion. All replicating sequence variants were separated by gel electrophoretic techniques and the sequences and their relative frequencies were determined. From both types of studies it can be concluded (i) that HP II is a functional element in the (-)strand replication intermediate, generated due to sequential folding during synthesis, and that it is essential for template activity of (+)strand synthesis; (ii) that G:U pairs are tolerated transiently in (-)strand HP II; the lower stability of such a HP II is compensated by additional mutations outside HP II which suppress the competition of a rod-like structure; and (iii) that the reversions are generated spontaneously during (-)strand synthesis. Furthermore, the double-stranded structure of HP II is the essential element for short term replication of PSTVd but the exact sequence of the wild-type proves to be superior with regard to fitness and replicability of PSTVd.
Base Sequence↗
Inhibition of potato spindle tuber viroid (PSTVd) infection with DNA oligonucleotides.
The effects of DNA oligonucleotides complementary to potato spindle tuber viroid (PSTVd) on viroid infection were investigated. The oligonucleotides were used to form hybrids with PSTVd in the infection mixture. A 75% reduction of viroid infection was found when an oligonucleotide complementary to nucleotides 79-110 of PSTVd was hybridized with PSTVd at a molar ratio of approximately 5000:1, respectively. A total inhibition of PSTVd infection was observed using an oligonucleotide complementary to nucleotides 42-78 at the same molar excess of DNA over PSTVd, although a 200-fold molar excess was found to be sufficient for the complete blocking of infection by PSTVd. This oligonucleotide caused a significant reduction (about 83%) of viroid infection even if the hybridization was done at a low (30 degrees C) temperature. Shorter oligonucleotides containing 22 and 15 bases corresponding to position 42-62 and 63-78, respectively, exhibited a significant effect only at a high (80 degrees C) initial temperature of molecular hybridization. Heteroduplexes formed between PSTVd RNA and antisense DNA were found to be less stable in a crude nuclease extract from tomato leaves as compared with PSTVd RNA alone. RNase H was demonstrated to cleave the molecular hybrids in vitro.
Base Sequence↗
Aneuploidy fraction but not DNA index is important for the prognosis of patients with stage I and II breast cancer--10-year results.
BACKGROUND: Individual assessment of the prognosis of patients with breast cancer is crucial for the selection of risk-adapted adjuvant therapy and in follow-up. Parameters from DNA flow-cytometry have been shown to provide significant prognostic information, but published results are in conflict and there are only a few investigations with long-term follow-up. The aim of this study is to clarify the impact of tumor DNA data on the clinical course of stage I and stage II breast cancer patients. PATIENTS AND METHODS: Several flow-cytometry DNA analyses were performed on tumor samples derived from 191 breast cancer patients entered in a controlled clinical trial after a median follow-up of more than 10 years. In addition to DNA index (DNI) and the percentage of cells in S phase (SPF), an index, designated aneuploidy fraction (AF), was determined. It ascertains the percentage of aneuploid cells out of all cells in the DNA flow-cytometry histogram, and its reproducibility has been tested by measurements of AF in two different samples of the same tumor. Univariate analyses and, in the 122 patients for whom complete information was available, a Cox model, were performed to investigate the individual prognostic impact of flow-cytometry parameters compared with established clinical factors. RESULTS: AF proved to be a very valuable prognostic indicator both in univariate and multivariate analyses, whereas DNI and SPF failed to provide independent prognostic information. The combination of AF and lymph node status clearly identifies different prognostic subgroups in operable breast cancer. CONCLUSIONS: Routine evaluation of patients with breast cancer should include tumor DNA flow-cytometry. Aneuploidy fraction is a valuable tool in assessing an individual patient's prognosis and thus can help in the choice of the appropriate adjuvant treatment strategy. Whether it, rather than DNI and SPF should be used, as we found, needs to be validated in a larger prospective investigation.
Aged↗
[Explanation of a pseudonym: for G. Steger it should be H.Grosse].
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Anonyms and Pseudonyms↗
Structural requirements for viroid processing by RNase T1.
Viroids are replicated via a rolling circle-like mechanism in which (+) strand oligomeric intermediates have to be cleaved enzymatically to unit-length molecules followed by ligation to mature circles. A transcript of potato spindle tuber viroid, which is still infectious, consists of a monomeric molecule with only 22 additional nucleotides, thus doubling part of the central conserved region of viroids. It was shown that this transcript can be cleaved and ligated in vitro to circles by RNase T1. To elucidate the site and mechanism of processing, 16 different site-specific mutants of this longer-than-unit-length transcript were constructed and analyzed by in vitro processing with RNase T1, infectivity studies, temperature-gradient gel electrophoresis, and structure calculations. The wild-type sequence and several mutated transcripts are able to adopt a particular secondary structure which is the prerequisite for enzymatic cleavage and ligation by RNase T1. This "processing structure" exposes both potential cleavage sites in the nearest spatial neighborhood, thus favoring the subsequent ligation to circles. Those mutated sequences for which the formation of the processing structure is impossible or thermodynamically highly unfavored are not processed. The results demonstrate that the particular structural features of viroids enable them to be cleaved and ligated by one and the same enzyme, RNase T1. The in vitro mechanism may serve as a mechanistic model for cellular processing of viroids.
Base Sequence↗
How do eukaryotic activator proteins stimulate the rate of transcription by RNA polymerase II?
A large number of activator proteins have now been identified in higher and lower eukaryotes, which bind to the regulatory regions of protein-encoding genes and increase the rate at which they are transcribed by RNA polymerase II. The mechanism by which activators function is being intensively studied and some of the targets of transcriptional activation domains have now been identified. These studies have also revealed novel classes of regulatory factors, which were not anticipated by extrapolating from the principles obtained with prokaryotic promoters.
Animals↗
Prognostic impact of karyotype and immunologic phenotype in 125 adult patients with de novo AML.
One hundred-twenty-five adult patients with de novo acute myeloid leukemia (AML) were treated according to a standard 7 + 3 induction regimen. Karyotype and immunological phenotype of blasts examined prior to treatment were correlated with each other, with response to treatment and duration of survival. The following monoclonal antibodies (mAbs) were used for immunological phenotyping: VIM-D5 (CD15), MY7 (CD13), MY9 (CD33), VIM-2 (CDw65), VIM-13 (CD14), 63D3 (CD14), VID-1 (anti HLA-DR), WT1 (CD7), CLB-Ery3 (antiblood group H antigen), C17-27 (CD61), and an antiserum against TdT. Despite a considerable overlap between the individual groups, patients with specific aberrations as defined by the MIC classification (n = 39) showed distinct, characteristic, myeloid or myelomonocytic immunophenotypes. In M2/t(8;21) there was a significant association with negativity to CD13, in M3/t(15;17) with negativity to CD15 and HLA-DR, whereas in M4/inv(16) expression of blood group H antigen was unexpectedly found. The response to therapy, as well as rate of complete remission as duration of survival, was better in patients with M2/t(8;21), M3/t(15;17), and M4Eo/inv(16) as compared to all other patients and significantly worse in patients with M5a/t/del(11)(q23). In 35 patients with normal karyotype and 16 patients with cytogenetic anomalies not presently associated with FAB subtypes the expected correlations of rather immature myeloid immunologic phenotypes with M1 and M2 morphology and CD14 expression in monoblastic leukemias was found. Remission rate and survival were significantly worse in 19 patients with complex nonrandom aberrations, where blast cell expression of blood group H antigen and of TdT were significantly increased.
Adolescent↗